Globins have evolved under strong selective pressure to overcome the natural tendency of a free heme prosthetic group to bind carbon monoxide (CO) 1000-fold more tightly than atmospheric dioxygen (O$\sb2$). Without protein-directed discrimination between these ligand molecules, the basal production of CO from various catabolic processes would inhibit the reversible transport and storage of oxygen that is necessary for life. Until recently, the exact function of active site residues in globin function has relied on studies of naturally occurring hemoglobin (Hb) mutants and globins from species with divergent active site residues.The desire to probe the role of Mb active site residues in ligand discrimination by manipulation of the protein at the genetic level inspired the total synthesis of a gene for sperm whale myoglobin. Total gene synthesis provided a system which circumvented the sometimes tedious cDNA gene cloning methods and some of the proposed problems associated with the expression of the cDNA clones for human hemoglobin chains and human myoglobin. When inserted into the plasmid pUC19, this construction produces soluble heme containing Mb to $\sim$10% of the total soluble E. coli cell protein. The high-level expression of authentic Mb in E. coli therefore provided the necessary starting point for site-directed mutagenesis efforts designed to probe the role of conserved active site globin residues.Replacement of His64, the distal histidine, with twelve different amino acids by site-directed mutagenesis revealed that virtually every amino acid placed in position 64 of the ligand binding pocket (with the possible exception of Gln) destroyed the ability of Mb to stabilize bound O$\sb2$ since the normal hydrogen bond donor had been removed.Site-directed mutagenesis of Val68 confirmed that this residue is also providing steric constraints on bound CO but not O$\sb2$, clearly indicating the concerted action of two active site residues in protein-ligand recognition. Replacement of Val68 with Phe resulted in an altered ligand pathway as evidenced by decreased O$\sb2$ association and dissociation rates with relatively unchanged CO rates.Comparison between the His64Gly Mb mutant and the analogous mutations in human Hb $\alpha$ and $\beta$ chains revealed that the Hb $\alpha$ chains are most like Mb, however the distal histidine in $\beta$ chains is essentially unimportant in R state hemoglobin ligand binding. (Abstract shortened with permission of author.)